Pectins are major constituents of the cell walls of edible parts of fruits and vegetables. The middle lamella which are situated between the cell walls are mainly built up from protopectin which is the insoluble form of pectin. Pectins are considered as intercellular adhesives and due to their colloid nature they also have an important function in the water-regulation of plants. Water-binding capacity is greatly increased by the amount of hydrophylic hydroxyl and carboxyl groups. The amount of pectin can be very high. For example, lemon peels are reported to contain pectin up to 30% of their dry weight, orange peels contain from 15-20% and apple peels about 10% (Norz, K., 1985. Zucker und Susswaren Wirtschaft 38 5-6).
Pectins are composed of a rhamno-galacturonan backbone in which 1,4-1-linked .alpha.-D-galacturonan chains are interrupted at intervals by the insertion of 1,2linked .alpha.-L-rhamnopyranosyl residues (Pilnik, W. and A. Voragen 1970. In `The Biochemistry of fruits and their products`, Vol. 1, Chapter 3, p.53. Acad. Press). Other sugars, such as D-galactose, L-arabinose and D-xylose, are present as side chains. A large part of the galacturonan residues is esterified with methyl groups at the C2 and C3 position.
Pectin-degrading enzymes are important tools in the food industry. Traditionally these enzymes are used as mixtures. Aspergillus niger and other fungi produce a whole spectrum of enzymes which can advantageously be used in the degradation of pectin. Examples of such enzymes are pectin esterase, pectin lyase (also called pectin transeliminase), endo- and exo-polygalacturonases. In A. niger the pectin degrading proteins are not expressed constitutively. Induction of these enzymes is achieved by growing the strains when carbon sources such as glucose or sucrose are limiting and in the presence of pectin or breakdown products thereof.
In order to avoid the problem of induction and also to avoid obtaining not well-defined enzyme mixtures there is a growing tendency to clone the genes encoding these enzymes and to express them in other more suitable host cells.
The cloning and expression of several of these enzymes obtained from Aspergillus niger has been reported. EP 0 278 355 describes the cloning of the pectin lyase gene, the sequence thereof and the expression. EP 0 353 188 adds some other pectin lyases.
As mentioned above pectin contains a backbone comprising a high amount of 1,4-linked .alpha.-D-galacturonan molecules. This backbone can be digested by the action of depolymerases. Two such depolymerases are known, endo- and exo-polygalacturonase. Examples of the cloning and expression of especially the former one of these depolymerases have been reported.
EP 0 421 919 discloses two polygalacturonases which can be classified as endo-polygalacturonases. Another endo-polygalacturonase has been disclosed in EP 0 388 593. Both of these patent application used Aspergilli the source of the gene.
Exo-polygalacturonases are not so abundant as the endo- form as evidenced by the reports about this enzyme published so far.
Mill (Biochem. J. 99: 557-561 and 562-565 (1966)) reported the isolation and characterization of two distinct exo-polygalacturonases. One of these was found to be mercury activated. This was confirmed by Hara et al. (Nippon Shokuhim Kogyo Gakkashi 31: 581-586 (1984)). Kester and Visser (Biotechn. Appl. Biochem, 12: 150-160 (1990)) report the presence of only one exo-polygalacturonase and 5 endo-polygalacturonases in A. niger culture filtrates. Finally, the cloning of exo-polygalacturonase from Erwinia chrysanthemi EC16 has been reported (He and Collmer J. Bacteriol. 172: 4988-4995 (1990)).
Exo-polygalacturonase is capable of converting polygalacturonides to galacturonic acid. To enhance this process it is advantageous to use pure exo-polygalacturonase. Galacturonic acid can be used as a source for different synthetic reactions it has for example been found to be useful in the production of ascorbic acid.